Brevibacterium frigoritolerans and microbial bacterial agent and their applicationTechnical field
The present invention relates to agricultural biological technical field, in particular it relates to a kind of brevibacterium frigoritolerans, Yi ZhongweiBacteria agent and their application.
Background technology
The disease that nematicide causes is to endanger one of corps diseases of being on the rise, agriculture of being typically injured in recent yearsCrop can underproduction 20-30%, serious underproduction degree can reach 60%, even have no harvest, extent of injury phaseWhen seriously.The line insect types that can result in harm has kind more than 3000, main line insect types to have southern root lineWorm, Java nematicide etc..Nematicide mainly with adult, ovum in diseased plant residuum or with larva in soil moreIn the winter, it also is able to survive under conditions of without host 3 years.In relatively good environment, ovum can be severalFirst-instar young is formed in hour.Second instar larvae is the most active, invades, mouth from host plant tip of a root children is tenderThe saliva of pin secretion induces killed cell transition to increase, and forms root nodule.Female male imago post-coitum, lay eggs inIn the cutin egg capsule at polypide rear portion, quantity is up to 100-300 grain.After next season of growth arrives, egg capsuleIn ovum can hatch growth, leave after two ages soil enter soil again infect harm.Nematicide adult is suitableTemperature conditions be 25-30 DEG C, temperature higher than 55 DEG C or less than 5 DEG C time, adult activity weakens.At 27 DEG CUnder conditions of, nematicide completes a generation needs 25-30 days, can occur in 1 year 5-8 generation.When spring, temperature was low," nematicide " morbidity evening, light, slow, after field planting one month, just see that fragmentary diseased plant occurs；Autumn stubble is cultivated,During temperature height, morbidity early, heavily, soon, can be fallen ill for after field planting 11 days.Nematicide is mainly distributed on 5-30In centimetre deep soil, but there is people to be even found that nematicide in the soil of 50 centimetres, add preventing and treatingDifficulty.
At present, the method for preventing and treating nematicide includes chemical medicinal method and measure of biotic control, and chemical prevention rule is such asWith Brom-O-Gas, the rich mu of dimension, must speed is gone out, lime nitrogen etc. carries out soil-fumigating, but this kind of soil fumigantThere is natural disposition of going out, the Pest organism Mus in soil and beneficial microbe etc. can be killed；Measure of biotic control such as makesKilling nematicide with nematicide biocontrol fungi, it is relatively low and be difficult to set up that nematicide biocontrol fungi is usually present germination rateFor controlling the population density required for nematode growth, them are caused to there is preventive effect on preventing and treating nematicide low and anti-The defect that effect is unstable.
Summary of the invention
The present inventor separated a strain brevibacterium frigoritolerans, finds that it has the nematicide of killing of excellenceEffect, resulting in the present invention.For the preventive effect overcoming the method for existing Biological control nematicide to existThe defect that low and preventive effect is unstable, the invention provides a kind of brevibacterium frigoritolerans, this brevibacterium frigoritoleransThe deposit number of (Brevibacterium frigoritolerans) is CGMCC NO.8837.
Present invention also offers a kind of microbial bacterial agent, this microbial bacterial agent contains thalline and culture medium, instituteState the brevibacterium frigoritolerans that thalline includes that deposit number is CGMCC NO.8837.
Present invention also offers brevibacterium frigoritolerans as above and microbial bacterial agent as above anti-Control the application in the disease of crops.
By technique scheme, the present invention can more efficiently prevent and treat root knot nematode disease and/or sporangiocystThe disease of the crops such as nematicide.
Other features and advantages of the present invention will be described in detail in detailed description of the invention part subsequently.
Biomaterial preservation
The brevibacterium frigoritolerans (Brevibacterium frigoritolerans) of the present invention is the invention of the present inventionThe pure culture that people separates from the scale biogas engineering biogas slurry sample that Yantai, Shandong gathers, its preservation is compiledNumber being CGMCC NO.8837, preservation date is on February 21st, 2014, and depositary institution is that China is micro-Biological inoculum preservation administration committee's common micro-organisms center, address is positioned at BeiChen West Road, Chaoyang District, BeiJing CityNo. 3 Institute of Microorganism, Academia Sinica of No. 1 institute, Classification And Nomenclature is brevibacterium frigoritolerans (Brevibacteriumfrigoritolerans)。
Detailed description of the invention
Hereinafter the detailed description of the invention of the present invention is described in detail.It should be appreciated that this place is retouchedThe detailed description of the invention stated is merely to illustrate and explains the present invention, is not limited to the present invention.
The invention provides a kind of brevibacterium frigoritolerans, this brevibacterium frigoritolerans (BrevibacteriumFrigoritolerans) deposit number is CGMCC NO.8837.
Present invention also offers a kind of microbial bacterial agent, this microbial bacterial agent contains thalline and culture medium, instituteState the brevibacterium frigoritolerans that thalline includes that deposit number is CGMCC NO.8837.
Wherein, the amount of the thalline contained in described microbial bacterial agent can in very large range change, such asIn every gram of described microbial bacterial agent, deposit number is the work of the brevibacterium frigoritolerans of CGMCC NO.8837Bacterium number can be (1-500) × 107CFU, under preferable case, in every gram of described microbial bacterial agent, preservation is compiledNumber be the viable count of the brevibacterium frigoritolerans of CGMCC NO.8837 can be (5-150) × 107CFU。
According to the present invention, the kind of described culture medium can in very large range change, can be by various energyIt is enough in the culture medium cultivating brevibacterium frigoritolerans, for example, it is possible to be beef-protein medium, meat soupThe conventional culture medium such as culture medium, LB culture medium are as seed culture medium, for the preservation of strain；And send outThe culture medium of ferment is generally used for producing, and the kind of these culture medium is the most known to those skilled in the art.Above-mentioned culture medium can be commercially available or according to " microbiological culture media handbook " (MicrobiologyCulture Media Manual) record prepare.Such as, seed culture medium can be Carnis Bovis seu Bubali cream eggWhite peptone culture medium, its contain the Carnis Bovis seu Bubali cream of 1-3g/L, the peptone of 5-15g/L, the sodium chloride of 3-8g/L,The agar of 15-20g/L.Such as fermentation medium can contain: the Semen Glycines powder of 40-150g/L, 40-150g/LStarch, the sodium chloride of 0.5-8g/L, the calcium carbonate of 0.5-5g/L, the potassium dihydrogen phosphate of 2-10g/L andThe ferrous sulfate of 1-10g/L.
Wherein, above-mentioned various culture medium are standby, such as after can carrying out sterilizing according to conventional sterilizing methodsSterilizing 10-30 minute under conditions of 115-125 DEG C and 1.5-2 normal atmosphere.
Wherein, the preparation method of described microbial bacterial agent may include that CGMCC NO.8837'sBrevibacterium frigoritolerans is inoculated in fermentation medium and cultivates.Under preferable case, make to obtain by cultivationIn every gram of described microbial bacterial agent, deposit number is the work of the brevibacterium frigoritolerans of CGMCC NO.8837Bacterium number can be (1-500) × 107CFU, more preferably (5-150) × 107CFU.Wherein, the temperature of cultivationDegree can be 28-30 DEG C, and the time of cultivation can be 16-25 hour.
Present invention also offers brevibacterium frigoritolerans as above and microbial bacterial agent as above anti-Control the application in the disease of crops.
It is particularly preferred that the disease of described crops includes nematicide.It is highly preferred that described nematicide bagInclude root knot nematode disease and/or cyst nematode disease.
Further describe the present invention by the following examples:
Embodiment 1
The present embodiment is for illustrating cultivation and the preparation of microbial bacterial agent of the brevibacterium frigoritolerans of the present invention.
By the brevibacterium frigoritolerans (Brevibacterium that deposit number is CGMCC NO.8837Frigoritolerans) (tryptone, the yeast of 5g/L containing 10g/L extract to be inoculated into LB culture mediumThing and the NaCl of 10g/L) in, cultivate to strain density OD under 30 DEG C and 120 revs/min vibrations600ValueFor 0.6-0.8, obtain seed bacterium solution.
By in above-mentioned for 100mL seed bacterium solution addition 100L fermentation medium, (fermentation medium contains 80g/LSemen Glycines powder, the starch of 80g/L, the sodium chloride of 3g/L, the calcium carbonate of 3g/L, the potassium dihydrogen phosphate of 5g/LFerrous sulfate with 5g/L), cultivate at 30 DEG C, be sampled in incubation and pass through microscopeDirect counting method is observed, until the viable count of the brevibacterium frigoritolerans in every gram of culture fluid is 108CFU,Obtained culture fluid is the microbial bacterial agent of the present embodiment.
Embodiment 2
Preparing microbial bacterial agent according to the method for embodiment 1, except for the difference that, fermentation medium used isSemen Glycines powder containing 60g/L, the starch of 100g/L, the sodium chloride of 2g/L, the calcium carbonate of 4g/L, 3g/LPotassium dihydrogen phosphate and the culture medium of ferrous sulfate of 3g/L.
Comparative example 1
By the article number purchased from ATCC it is25097TMBrevibacterium frigoritolerans (BrevibacteriumFrigoritolerans) (tryptone, the yeast of 5g/L containing 10g/L extract to be inoculated into LB culture mediumThing and the NaCl of 10g/L) in, cultivate to strain density OD under 30 DEG C and 120 revs/min vibrations600ValueFor 0.6-0.8, obtain seed bacterium solution.
By in above-mentioned for 100mL seed bacterium solution addition 100L fermentation medium, (fermentation medium contains 80g/LSemen Glycines powder, the starch of 80g/L, the sodium chloride of 3g/L, the calcium carbonate of 3g/L, the potassium dihydrogen phosphate of 5g/LFerrous sulfate with 5g/L), cultivate at 30 DEG C, be sampled in incubation and pass through microscopeDirect counting method is observed, until the viable count of the brevibacterium frigoritolerans in every gram of culture fluid is 108CFU,Obtained culture fluid is the microbial bacterial agent of this comparative example.
Testing example 1
In this testing example, in laboratory environment testing example 1,2 and the microorganism of comparative example 1The microbial inoculum inhibitory action to Meloidogyne incognita (Meloidogyne incognita).Meloidogyne incognita is rootAccording to document (plant root-knot nematode biology, taxonomic identification and preventing and treating. Beijing: Science Press, 1983) inMethod, from the old complaint of the Fructus Lycopersici esculenti infected by Meloidogyne incognita gather.Concrete acquisition method bagInclude: the Fructus Lycopersici esculenti old complaint clear water suffering from nematicide is cleaned, with tweezers choose in old complaint fresh, full,Ripe pieces of an egg.Pieces of an egg are placed on equipped with (every gram of pieces of an egg water 5mL) in the culture dish of sterilized water, 25 DEG CCalorstat is cultivated, collected once the Meloidogyne incognita second instar larvae of new hatching every 24 hours, andNematicide is suspended in normal saline the concentration reaching 500/milliliter, stand-by as nematicide suspension.
The microbial bacterial agent of embodiment 1,2 and comparative example 1 is separately added in nematicide suspension as placeReason experiment, adds 1g microbial bacterial agent in every 20mL nematicide suspension, and with blank fermentation culture(fermentation medium contains the Semen Glycines powder of 80g/L, the starch of 80g/L, the sodium chloride of 3g/L, 3g/L to baseCalcium carbonate, the potassium dihydrogen phosphate of 5g/L and the ferrous sulfate of 5g/L) as blank, 25 DEG C of constant temperatureAfter case is cultivated 1 day, under microscope, living nematodes is counted, the verge of death borer population of experiment will be processedDivided by the verge of death borer population of blank and be scaled percent, obtain nemic death rate, result such as table 1Shown in.
Table 1
| Nemic death rate (%) |
| Embodiment 1 | 85.5 |
| Embodiment 2 | 83.1 |
| Comparative example 1 | 3.0 |
Data according to table 1 are visible, and the deposit number of the present invention is the cold-resistant of CGMCC NO.8837Brevibacterium (Brevibacterium frigoritolerans), relative to other brevibacterium frigoritolerans, has prominentThe ability killing nematicide.
Testing example 2
This testing example, by using in tomato green plantation, measures embodiment 1,2 and comparative example 1The microbial bacterial agent prevention effect to nematicide.
According to document (plant root-knot nematode biology, taxonomic identification and preventing and treating, Beijing: Science Press,1983) method in, gathers Meloidogyne incognita and infects the tomato plant of chamber planting.Concrete behaviourWork includes: cleaned by the Fructus Lycopersici esculenti old complaint clear water suffering from nematicide, chooses in old complaint fresh, full with tweezersFull, ripe pieces of an egg.Pieces of an egg are placed on equipped with (every gram of pieces of an egg water 20mL) in the culture dish of sterilized water,Crushing ovum grain gently so that line eggs is dispersed in water, stand-by as line eggs grain suspension.Treat Fructus Lycopersici esculentiWhen there is the first fringe fruit, at the root soil of every strain Fructus Lycopersici esculenti, the place of burying instills 10mL line eggs grain suspension,After 10 days, there is the disease of nematicide in the root of tomato plant.
In different growing areas, to occur nematicide disease (after spraying pathogenic bacterium solution 10 days) kindEggplant plant carries out pouring root and uses embodiment 1,2 and the microbial bacterial agent of comparative example 1, and amount of application is 5g/Strain, with fermentation medium, (fermentation medium contains the Semen Glycines powder of 80g/L, the starch of 80g/L, 3g/LSodium chloride, the calcium carbonate of 3g/L, the potassium dihydrogen phosphate of 5g/L and the ferrous sulfate of 5g/L) substitute micro-lifeThing microbial inoculum is as space management, in addition to using microbial bacterial agent by such scheme, and other kind of each growing areaPlant condition identical.It is 3 days that pouring root uses the interval of microbial bacterial agent, and continuous pouring root is used 3 times,Within 10 days, 20 days and 30 days, investigate the state of an illness respectively after last dispenser, in units of strain, carry out classification noteCarrying, the degree of root-knot nematode harm raw situation with root knot and is represented, raw many major generals state of an illness according to root knotIt is divided into 5 grades: 0 grade: without root knot；1 grade: root knot accounts for the 1%-25% of full root system；2 grades: root knot accounts for entirelyThe 26%-50% of root system；3 grades: root knot accounts for the 51%-75% of full root system；4 grades: root knot accounts for full root system76%-100%.The Fructus Lycopersici esculenti nematicide state of an illness of growing area is investigated and counted, based on formula (A)Calculating disease index, calculate prevention effect according to formula (B), result is as shown in table 2.
Table 2
From the data of table 2 it can be seen that the microbial bacterial agent of the present invention has excellent to Fructus Lycopersici esculenti nematicidePrevention effect.
The preferred embodiment of the present invention described in detail above, but, the present invention is not limited to above-mentioned realityExecute the detail in mode, in the technology concept of the present invention, can be to the technical side of the present inventionCase carries out multiple simple variant, and these simple variant belong to protection scope of the present invention.
It is further to note that each the concrete technology described in above-mentioned detailed description of the invention is specialLevy, in the case of reconcilable, can be combined by any suitable means, in order to avoid need notThe repetition wanted, various possible compound modes are illustrated by the present invention the most separately.
Additionally, combination in any can also be carried out between the various different embodiment of the present invention, as long as itsWithout prejudice to the thought of the present invention, it should be considered as content disclosed in this invention equally.